Pluripotent stem cells, due to their differentiation pluripotency capable of being differentiated into any tissue, are widely used in various fields such as a tissue differentiation study, a drug test, a regenerative medicine and the like. In particular, since the establishment of iPS cells, the development of research in this field is remarkable. A variety of efforts for realization of a regenerative medicine has been made all over the word.
However, the pluripotent stem cells are easily differentiated and may possibly lose pluripotency once they are differentiated. Thus, the culture of the pluripotent stem cells needs to be performed while maintaining an undifferentiated state of the pluripotent stem cells. It can be said that the maintenance of the undifferentiated state is one of the most important factors in the culture of the pluripotent stem cells.
In order to maintain the undifferentiated state, the use of a differentiation-inhibiting agent or the removal of differentiation-started pluripotent stem cells is carried out. In the mass production of pluripotent stem cells, one of the problems which may cause the most significant obstacle is the removal of differentiation-started pluripotent stem cells. If the removal of differentiation-started cells is insufficient, there is a possibility that the differentiation of surrounding cells is induced, thereby adversely affecting all the cells under culture. However, it is difficult for an unskilled technician to determine whether pluripotent stem cells are in an undifferentiated state. Under these circumstances, there is naturally a limit in the mass production of pluripotent stem cells. Thus, there is a demand for the development of a method for confirming whether pluripotent stem cells are in an undifferentiated state without relying on at least the judgment of a skilled technician or the development of a method for automatically determining differentiation-started pluripotent stem cells.
Previously, a qRT-PCR method, an immunostaining method or a flow cytometry method has been used as a method for confirming whether pluripotent stem cells are in an undifferentiated state (Non-patent Document 1). However, in the qRT-PCR method and the immunostaining method, it is necessary to destroy cells during measurement. In the flow cytometry method, it is possible to perform non-destructive measurement. However, it is necessary to suspend cells in a single cell state, which requires a complicated operation.
In the meantime. Patent Document 1 discloses a method for non-invasively determining the degree of stratification of cultured cells. However, this method is a method for determining the degree of stratification of cultured cells through the comparison with a predetermined threshold value.
Patent Document 2 discloses a method for determining the degree of stratification and/or differentiation of cultured cells. However, this method is a method for determining the degree of stratification and/or differentiation of cultured cells through the reference to a predetermined correlation database.
Accordingly, a demand still exists for the development of a non-destructive simple method as a method for confirming an undifferentiated state of pluripotent stem cells.